The invention relates to solvents useful for oligonucleotide synthesis and to the use of such solvents for assembling an array of oligonucleotides on a support surface.
The genetic information generated by the Human Genome Project is allowing scientists, physicians, and others to conduct diagnostic and experimental procedures on an unprecedented scale in terms of speed, efficiency, and number of screenings performed within one procedure. In order to make full use of the new information, there is an urgent need for the ability to screen a large number of oligonucleotide probes against samples of DNA or RNA from normal or diseased cells and tissue. One important tool for such analyses is nucleic acid hybridization, which relies on the difference in interaction energies between complementary versus mismatched nucleic acid strands. Using this tool, it is possible to determine whether two short pieces of nucleic acid are exactly complementary. Longer nucleic acids can also be compared for similarity.
Nucleic acid hybridization is often used in screening cloned libraries to identify similar, thus presumably related, clones. This procedure typically involves natural nucleic acid targets which are usually bound to a membrane, and a natural or synthetic nucleic acid probe, which is washed over many targets at once. With the appropriate mechanics, membranes can be constructed with targets at a density of between one and ten targets per mm.sup.2. Hybridization detection is carried out by labeling the probe, either radioactively or with chemiluminescent reagents, then recording the probe's emissions with film.
Alternative approaches to that described above for nucleic acid hybridization have attempted to employ an array of oligonucleotide probes synthesized on a solid support and then hybridized to a single natural target. These alternative approaches have yielded systems for assembling an array of oligonucleotides on a large scale, but the cost of making a variety of arrays is prohibitive.
For example, Brennan, U.S. Pat. No. 5,474,796, describes a piezoelectric impulse jet pump apparatus for delivering oligonucleotide synthesis reagents to array plates. The array plate is held in a mechanical stage that can be moved along the X and Y directions to position the plate under the appropriate jet nozzle. A separate nozzle head is provided for each of the four nucleotide monomers, and a fifth head delivers an activating reagent for synthetic coupling. Brennan describes that the solvents of choice for synthetic coupling in the jet pump apparatus are acetonitrile and diethylglycol dimethyl ether. Successful operation of the apparatus is not shown since the patent contains no data on the synthesis and assembly of multiple oligonucleotides.
Baldeschwieler (WO 95/25116) similarly describes an automated system for delivering oligonucleotide synthesis reagents to a substrate on which an array of oligonucleotides is produced. The patent envisions a five jet system, with one jet for each of the four nucleotide reagents, and one jet for the activating tetrazole solution. The movement of the device across the X and Y axis is computer-controlled, with the ink jet delivering reagent upwards to the underside of a microscope slide. According to Baldeschwieler, suitable solvents for the automated system include dibromomethane, nitromethane, acetonitrile, and dimethyl formamide. The deprotection reagent is 0.8 M ZnBr2 in 9:1 nitromethane:isopropanol.
Baldeschwieler also provides a description of the use of the ink jet system to synthesize an oligonucleotide. However, in contrast to the envisioned automated system, the ink jet was only used for delivery of the deprotecting agent. The 4.times.5 arrays of poly-T oligonucleotides were instead synthesized using a standard phosphoramidite synthetic cycle. The slide was exposed to the phosphoramidite monomers dissolved in acetonitrile in a reaction trough. Successive cycles of deprotection were carried out using the ink jet nozzle to apply the deprotection agent. However, the phosphoramidite monomers were added by exposing the slide to reaction solution in a trough, not by application using the ink jet nozzles.
Thus, automated application of nucleotides in solution to specific addresses on a matrix, using an ink jet nozzle system was not demonstrated by Brennan and/or by Baldeschwieler. There is no evidence that either system was operational for its intended purpose. In each case acetonitrile was a preferred solvent for nucleotide phosphoramidites, but in Baldeschwieler's working example, the matrix was exposed to the nucleotides in acetonitrile in a trough, not by ink jet application.
The solvent acetonitrile has several disadvantages that render it unsuitable for use in these automated systems. Acetonitrile has a low boiling point (81.degree. C.) and evaporates quickly at room temperature. This causes problems with the drops evaporating before the necessary chemical reactions have gone to completion. Evaporation of acetonitrile at the nozzle causes the solutes to crystallize out and clog the nozzle. Acetonitrile also has a low surface tension of about 29 dynes per cm. This low surface tension causes it to wet the face of the nozzle and leads to unstable drop formation. Also, acetonitrile is incompatible with several materials, such as glues and plastics, used in commercial ink-jet printer heads.
Thus, there exists a need for a method of synthesizing an array of oligonucleotides on a solid support, wherein the oligonucleotides remain bound to the support, or are removed for subsequent applications. In particular, there exists a need for a solvent suitable for use in the automated synthesis of the oligonucleotide arrays. The present invention satisfies this need and provides related advantages as well.